1. Field of the Invention
This invention relates to biogenic amine receptors from mammalian species and the genes corresponding to such receptors. Specifically, the invention relates to the isolation, cloning and sequencing of complementary DNA (cDNA) copies of messenger RNA (mRNA) encoding a novel mammalian biogenic amine receptor gene. The invention also relates to the construction of recombinant expression constructs comprising cDNA of this novel receptor gene, said recombinant expression constructs being capable of expressing receptor protein in cultures of transformed prokaryotic and eukaryotic cells. Production of the receptor protein in such cultures is also provided. The invention relates to the use of such cultures of such transformed cells to produce homogeneous compositions of the novel biogenic amine receptor protein. The invention also provides cultures of such cells producing this receptor protein for the cation of novel and useful drugs. Antibodies against and epitopes of this novel biogenic amine receptor protein are also provided by the invention
2. Background of the Invention
Biogenic amines are a class of naturally-occurring amino acid derivatives having a variety of physiological effects in the peripheral and central nervous systems. The parent compound is β-phenylethylamine, and derivatives of this compound include the biogenic amines. The biogenic amines are a large and diverse class of compounds that include dopamine, noradrenaline, epinephrine, norepinephrine, and serotonin. The biogenic amines are implicated in a variety of psychiatric and neurologic disorders.
In the periphery, biogenic amines are released by the sympathetic nervous system and adrenal medulla and are involved in integrating physiological responses to stress, while in the central nervous system the biogenic amines constitute some of the most important neurotransmitter systems.
The effects of biogenic amines are mediated through their receptors and their associated cell signaling systems (reviewed in Hoffmian & Lelkowitz, 1982, Ann Rev. Physiol. 44: 475-484; Civelli et al., 1993, Ann. Rev. Pharm. & Tox. 33: 281-307). These receptors are located in the plasma membrane of biogenic amine-sensitive cells. Structurally, they are characterized by having a pattern of seven transmembrane domains (see, for example, U.S. Pat. Nos. 5,422,265, 5,569,601, 5,594,108, 5,883,226, 5,880,260, 5,427,942 and 5,686,573). Functionally, certain of these receptors interact with adenylate cyclase, either stimulating or inhibiting the production of cyclic AMP thereby. These receptors include the adrenergic receptors (the a-1, a-2, b-1, b-2, and b-3 adrenergic receptors) and the dopanine receptors (the D1-, D2-, D3-, D−4-, and D5-dopamine receptors).
For example, epinephrine (adrenaline) and norepinephrine, as well as synthetic agonists of these biogenic amines which mimic their biological functions, and antagonists which block these biological functions, exert their effects by binding to specific recognition sites, (membrane receptors) situated on the cell membranes in the nervous system. Two principal classes of adrenergic receptors have been defined, the alpha-adrenergic receptors and the beta-adrenergic receptors. Five subtypes of adrenergic receptors (a-1, a-2, b-1, b-2, and b-3 adrenegic receptors) have now been distinguished. The genes encoding these receptors have been isolated and identified (Cotecchia et al., 1988, Proc. Natl. Acad. Sci. USA 85: 7159-7163; Kobilka et al., 1987, Science 238: 650-656; Frielle et al., 1987, Proc. Natl. Acad. Sci. USA 84: 7920-7924; Emorine et al., 1987, Proc. Natl. Acad. Sci. USA 84: 6995-6999; Eorine et al., 1989, Science 245: 1118-1121). Analysis of these genes has made it possible to recognize that they belong to a family of integral membrane receptors exhibiting some homology (Dixon et al., 1998, Annual Reports in Medicinal Chemistry, 221-223; Emorine et al., 1988, Proc. NATO Adv. Res. Workshop), especially at portions of the seven transmembrane regions that are coupled to regulatory proteins, called G proteins, capable of binding molecules of guanosine triphosphate (GTP).
These membrane receptors, after they have bound the appropriate ligand (agonist or antagonist), are understood to undergo a conformational change that induces an intracellular signal that modifies the behavior of the target cell. Beta-adrenergic receptors catalyze the activation of a class of G proteins, which in turn stimulates the activity of adenylate cyclase when they bind with biogenic amine agonists, whereas alpha-adrenergic receptor antagonists act in competition with the agonists for the binding to the receptor and prevent the activation of adenylate cyclase. When adenylate cyclase is activated, it catalyses the production of an intracellular mediator or second messenger, especially cyclic AMP.
In the central nervous system, dopamine is a biogenic amine neurotransmitter that modulates neuronal cells involved in movement initiation, appetitive behavior, hormone release, and visual dark adaptation. In the periphery dopamine plays a role in modulating blood pressure and renal function (see generally Cooper et al., 1978, THE BIOCHEMICAL BASIS OF NEUROPHARMACOLOGY, 3d ed., Oxford University Press, New York, pp, 161-195). The diverse physiological actions of dopamine are in turn mediated by its interaction with family of distinct dopamine receptors subtypes that are either “D1-like” or “D2-like,” which respectively stimulate and inhibit the enzyme adenylate cyclase Kebabian & Calne, 1979, Nature 277: 93-96). Alterations in the number or activity of these receptors may be a contributory factor in disease states such as Parkinson's disease (a movement disorder) and schizophrenia (a behavioral disorder) and attention deficit hyperactivity disorder (ADHD).
A great deal of information has accumulated regarding the biochemistry of the D1 and D2 dopamine receptors, and methods have been developed to solubilize and purify these receptor proteins (see Senogles et, al, 1986, Biochemistry 25: 749-753; Sengoles et al., 1988, J. Biol. Chem. 263: 18996-19002; Gingrch et al., 1988, Biochemistry 27: 3907-3912). The D1 dopamine receptor in several tissues appears to be a glycosylated membrane protein of about 72 kD (Amlaiky et al., 1987, Mol. Pharmacol. 31: 129-134; Ninzik et al., 1988, Biochemistry 27: 7594-7599). The D2 receptor can also be glycosylated and has been suggested to have a higher molecular weight of about 90-150 kD (Amlaiky & Caron, 1985, J. Biol. Chem. 260: 1983-1986; Amlaiky & Caron, 1986, J. Neurochem. 47: 196-204; Jarvie et al., 1988, Mol. Pharmacol. 34: 91-97).
Dopamine receptors are primary targets in the clinical treatment of psychomotor disorders such as Parkinson's disease and affective disorders such as schizophrenia (Seeman et al., 1987, Neuropsychopharm. 1: 5-15; Seeman, 1987, Synapse 1:152-333). Five different dopamine receptor genes (1, D2, D3, D4 and D5) and various splice variants of their transcripts have been cloned as a result of nucleotide sequence homology which exists between these receptor genes (Bunzow et al., 1988, Nature 336: 783-787; Grandy et al., 1989, Proc. Natl. Acad. Sci. USA 86: 9762-9766; Dal Toso et al., 1989, EMBO J. 8: 4025-4034; Zhou et al., 1990, Nature 346: 76-80; Sunahara et al., 1990, Nature 346: 80-83; Sokoloff et al., 1990, Nature 347: 146-151; Civelli et al., 1993, Annu. Rev. Pharmacol. Toxicol. 33: 281-307; Van Tol et al., 1991, Nature 350: 610-4).
Biogenic amine receptors are also targets for a host of therapeutic agents for the treatment of shock hypertension, arrhythmias, asthma, migraine headache, and anaphylactic reactions, and include antipsychotic drugs that are used to treat schizophrenia and β-blockers used to control high blood pressure.
In addition to these compounds, a number of biogenic amines are present in much lower quantities (less than 1% of the biogenic amines) and are therefore known as trace amines. The trace amines include such compounds as para-tyramine (p-tyramine), meta-tyramine (m-tyramine), phenylethylamine, octopamine, and tryptamine. The trace amines β-phenethylamine (β-PEA), p-tyramine, tryptamine, and octopamine are found in peripheral tissues as well as the central nervous system (Tallman et al., 1976, J Pharmacol Exp Ther 199: 216-221; Paterson et al., 1990, J Neurochem 55: 1827-37). In vivo β-PEA and p-tyramine can be synthesized from phenylalanine or tyrosine by the enzyme amino acid decarboxylase. (Boulton and Dyck, 1974, Life Sci 14: 2497-2506; Tallman et al., 1976, ibid.).
Investigations into the effects of trace amines on smooth muscle and glandular preparations early in the twentieth century clearly demonstrated that amines produced by putrefaction and lacking the catechol nucleus were capable of producing robust sympathomimetic effects (Barger and Dale, 1910, J Physiol 41: 19-59). Currently it is thought that p-tyramine and β-PEA manifest their peripheral effects by promoting the efflux of catecholamines from sympathetic neurons and adrenals (Schonfeld and Trendelenburg, 1989, Naunyn Schmiedeberg's Arch Pharmacol 339: 433-440; Mundorf et al., 1999, J Neurochem 73: 2397-2405) which results in the indirect stimulation of adrenergic receptors (Black et al., 1980, Eur J Pharmacol 65: 1-10).
Sensitive techniques have been developed to detect low concentrations of trace amines in the central nervous system. Such studies have revealed that trace amines in the central nervous system have a high turnover rate (Meek et al., 1970, J. Neurochem. 17: 1627-1635; Lemberger et al., 1971, J. Pharmac. Exp. Ther. 177:169-176; Wu & Boulton, 1974, Can. J. Biochem. 52:374-381; Durden & Philips, 1980, J. Neurochem. 34: 1725-1732). Trace amines are expressed throughout the brain in a heterogenous pattern and at least two of them can pass easily across the blood-brain-barrier (Boulton, 1974, Lancet ii: 7871; Oldendorf, 1971, Am. Physiol. 221: 1629-1639). Trace amines are also known to potentiate caudate neuronal firing in response to dopamine application and act as sympathomimetics by stimulating release of biogenic amines from brain preparations and synaptosomes when applied at high concentrations. Para-tyramine may act as a “false transmitter” in a manner similar to that of amphetamine by triggering release of neurotransmitters such as dopamine.
The abilities of p-tyramine and β-PEA to deplete neurotransmitter from storage vesicles, compete with neurotransmitters for uptake, and stimulate outward neurotransmitter flux through the plasma membrane carders are similar to the actions of the β-PEA analog, α-methyl-β-phenethylamine, better known as amphetamine (Amara and Sonders, 1998, Drug Alcohol Depend 51:87-96; Seiden et al., 1993). Amphetamines were originally marketed as stimulants and appetite suppressants, but their clinical use is now mostly limited to treating attention deficit hyperactivity disorder (Seiden et al., 1993, Annu Rev Pharmacol Toxicol 33:639-677): Although listed as controlled substances, amphetamines are widely consumed because of their ability to produce wakefulness and intense euphoria. Some substituted amphetamines, such as MDMAN (“ecstasy”) and DOI, are taken for their “empathogenic” and hallucinogenic effects. (Eisner, 1994, Ecstasy: The MDMA story. Ronin Books, Berkeley, Calif.; Shulgin and Shulgin, 1991, PiHKAL: A chemical love story. Transform Press, Berkeley, Calif.). Numerous liabilities are associated with the use of amphetamines including hyperthermia (Byard et al., 1998, Am J Forensic Med Pathol 19:261-265), neurotoxicity (Ricaurte and McCann, 1992, Ann NY Acad Sci 648:371-382), psychosis (Seiden et al., 1993, ibid.), and psychological dependence (Murray, 1998, J Psychol 132: 227-237). In addition to the actions of amphetamines at biogenic amine transporters, it is also clear that a subset of amphetamine analogs, especially those with hallucinogenic properties, can act directly on 5-HT receptors as they have much higher affinities for these sites than for the transporters (Marek and Aghajanian, 1998, Drug Alcohol Depend 51:189-198).
The importance of biogenic amines and their receptors, particularly in the brain and central nervous system, has created the need for the isolation of additional biogenic amine receptors, particularly trace amine receptors, for the development of therapeutic agents for the treatment of disorders, including disorders of the CNS and most preferably treatment of disorders on mental health such as psychosis, in which biogenic amines and their receptors have been implicated. There is also a need for developing new tools that will permit identification of new drug lead compounds for developing novel drugs. This is of particular importance for psychoactive and psychotropic drugs, due to their physiological importance and their potential to greatly benefit human patients treated with such drugs. At present, few such economical systems exist. Conventional screening methods require the use of animal brain slices in binding assays as a first step. This is suboptimal for a number of reasons, including interference in the binding assay by non-specific binding of heterologous (i.e., non-receptor) cell surface proteins expressed by brain cells in such slices; differential binding by cells other than neuronal cells present in the brain slice, such as glial cells or blood cells; and the possibility that putative drug binding behavior in animal brain cells will differ from the binding behavior in human brain cells in subtle but critical ways. The ability to synthesize human biogenic amine receptor molecules in vitro would provide an efficient and economical means for rational drug design and rapid screening of potentially useful compounds. For these and other reasons, development of in vitro screening methods for psychotropic drugs has numerous advantages and is a major research goal in the pharmaceutical industry.